Cancer is widely recognized as one of the major challenges to the healthcare industry, in terms of the variety of specific disease processes embraced by the term, the number of people and animals afflicted, and the effort and resources devoted to its treatment. For years, cancer has resisted man's attempts to understand and, hence, control the disease. Although that resistance has been overcome in certain contexts, the major, broad-based therapeutic approaches to cancer treatment continue to be burdened by deleterious side effects. For example, chemotherapy involves the delivery of cytotoxic compounds that target dividing cells, thus preferentially, but imperfectly, destroying cancer cells. Healthy dividing cells are also lost, however, and the treatments can lead to serious, life-threatening complications and the treatments frequently result in pain, nausea, hair loss, and a highly increased risk of serious infection. Radiotherapy, another broad-based approach, also exhibits imperfect targeting of cancer cells, with the result that healthy as well as cancerous cells can receive a lethal dose of radiation, leading to side effects such as pain, loss of vigor, and an increased risk of secondary malignancies, up to 20%, in some cases.
By way of example, ovarian carcinoma represents a significant women's health concern, as it is the most common cause of death from gynecological malignancy in the Western world (1). Within the spectrum of ovarian carcinomas, surface epithelial tumors represent 90% of all malignant ovarian neoplasms (2). Survival rates for surface epithelial ovarian carcinoma (30-40%) have remained relatively constant for the past 30 years (1), primarily due to the fact that metastatic spread via the lymphatics and by peritoneal implantation is clinically silent, resulting in a late stage at presentation.
Despite the abundance of molecular studies in the field of cancer research, significant independent prognostic indicators used in treatment stratification of patients with ovarian tumors are primarily clinical. They include age at diagnosis, International Federation of Gynecology and Obstetrics (FIGO) stage at presentation, and residual disease after surgery. Common molecular abnormalities described in ovarian tumors include mutations in the TP53 tumor suppressor gene, genetic amplification of the growth factor Her-2/neu (c-erbB-2), and loss of the distal half of chromosome 1 Sq (3-6). The expression of Survivin has also recently been demonstrated to be aberrantly elevated in over 70% of epithelial ovarian tumors (7, 8).
Ovarian carcinoma is the fifth leading cause of death from cancer among women in the United States, and the fourth among women over 40 years of age, resulting in an estimated 14,000 deaths per year (1). Although treatment of early stage ovarian cancer yields 5-year survival rates close to 90% (39), approximately 25-40% of patients (especially those with unfavorable prognostic indicators) are likely to relapse. Patients who clinically relapse less than 6 months after chemotherapy have very limited treatment options, often with low response rates to standard chemotherapeutic agents and a poor median survival (11 months) (9). For this reason there is a pressing need for the development of novel therapies that will effectively treat advanced and recurrent ovarian carcinoma.
The immune system also plays a role in combating cancer. CTL-mediated immunity is an important natural response to tumor cell growth (11, 40). It is also an important therapeutic avenue that has been explored in clinical trials to reduce tumor cell proliferation (41-43). Most immunotherapy studies targeted to cancer cells rely on the use of activated T-lymphocytes to perform this action. These studies can be hampered by the lack of antigen-presenting tumor cells within the patient. Suicide gene approaches are currently being tried by a number of different groups to treat cancer (44, 45), but specificity and efficacy concerns remain.
Successful treatment of ovarian cancer ultimately depends on clinical response to therapy. Early-stage ovarian cancer is most sensitive to platinum-based chemotherapy regimens, which have been the gold standard in the treatment of this disease (9). The preferred therapeutic regimen for advanced stage ovarian carcinoma relies on a combination of a platinum-based compound (cisplatin or carboplatin) and a taxane (commonly paclitaxel) (10). Treatment of advanced stage and recurrent ovarian carcinoma is frequently hampered by high rates of chemoresistance. Research on the development and efficacy of therapeutics for ovarian carcinoma is critical to improve patient survival. Accordingly, the medical and veterinary communities continue to seek treatment modalities that will provide better targeting of cancer cells with a capacity to deliver an efficacious dose of cytotoxin to such cells.
Molecular biology has been contributing significant advances to health care for several decades. Although early efforts to harness recombinant DNA technologies for use in health care were occasionally problematic, the past decade has seen a dramatic increase in the reliability and efficacy of recombinant DNA methodologies used to provide health care. Today, man's understanding of the processes controlling gene expression has developed to the point where the medical and veterinary communities are receptive to this approach to the treatment and amelioration of a wide variety of conditions and diseases.
Programmed cell death (also referred to as apoptosis) is distinguishable, both morphologically and functionally, from necrosis. Programmed cell death is a natural form of death that organisms use to dispose of cells. Cells dying by programmed cell death usually shrink, rarely lyse, and are efficiently removed from the organism (rapidly recognized and engulfed by macrophages) without the appearance of inflammation.
Apoptosis was initially used to describe a subset of programmed cell deaths sharing a particular set of morphological features that include membrane blebbing, shrinkage of cytoplasm, chromatic condensation and formation of a “DNA ladder” (i.e., DNA fragmentation). During apoptosis, cells lose their cell junctions and microvilli, the cytoplasm condenses, and nuclear chromatin marginates into a number of discrete masses. While the nucleus fragments, the cytoplasm contracts and both mitochondria and ribosomes become densely compacted. After dilation of the endoplasmic reticulum and its fusion with the plasma membrane, the cell breaks up into several membrane-bound vesicles, referred to as apoptotic bodies, which are usually phagocytosed by adjacent cells. Activation of particular genes, such as tumor suppressor genes in vertebrates, is thought to be necessary for apoptosis to occur. Apoptosis induced by numerous cytotoxic agents can usually be suppressed by expression of the anti-apoptotic gene bcl-2, which produces a cytoplasmic protein, Bcl-2.
Survivin has recently been identified as an inhibitor of apoptosis protein (IAP), a relatively small group of related proteins that inhibit the apoptotic process by interfering with caspase function. The first IAP was discovered in baculovirus and IAPs have now been reported in Drosophila, chick, mouse and human. Five human IAPs have been identified: HIAP1, HIAP2, XIAP (X-chromosome linked IAP), NAIP (neuronal apoptosis inhibiting protein) and Survivin. The gene encoding human Survivin is located on chromosome 17q25. Survivin is a 16.5 kD protein originally identified as cytoplasmic, but now known to be present in the nucleus and mitochondria as well. Survivin contains a single partially conserved BIR domain, and a highly charged carboxyl-terminus coiled-coil region instead of a RING finger, which inhibits apoptosis induced by growth factor withdrawal, UV-irradiation, Fas ligand, and other pro-apoptotic stimuli.
The Survivin promoter has been shown to be relatively silent in non-malignant cells and tissues both in vitro and in vivo (23, 24). Furthermore the survivin gene is relatively silent in non-transformed, differentiated cells (25). Expression of Survivin occurs in G2/M in a cell cycle-dependent manner, and the gene product localizes to mitotic spindle microtubules and intercellular acto-myosin bridges, i.e., midbodies, during cell division. Interference with this topography, or blocking survivin expression, caused increased caspase-3 activity in G2/M and a profound dysregulation of mitotic progression. Remarkably, Survivin was identified as one of the top four “transcriptomes,” out of 3.5 million mRNAs uniformly expressed in cancer but not in normal tissues. Additionally, it has been shown that transformed cells are exquisitely sensitive to manipulation at this mitotic stage as interference with Survivin expression and function using dominant-negative mutants with point mutations in the conserved baculovirus IAP repeat (BIR) domain, or survivin antisense, resulted in aberrant mitoses and spontaneous apoptosis.
Unlike other members of the IAP family, Survivin has only one BIR domain and does not have a carboxy-terminal RING finger. Instead, Survivin has a carboxy-terminal coiled-coil region. Based on overall sequence conservation, the absence of a carboxy terminus RING finger and the presence of a single, partially conserved, BIR domain, Survivin shares the highest degree of similarity with its C. elegans and yeast orthologs. Importantly, Survivin is minimally expressed in adult tissues, but is prominently expressed in most common human cancers, including cancers of the lung, colon, breast, pancreas, prostate, and central nervous system, and in about 50% of high-grade non-Hodgkin's lymphomas. For example, Survivin has been detected in adenocarcinoma of the pancreas, breast adenocarcinoma, colon cancer, head and neck squamous cell carcinoma, neuroblastoma, malignant thymoma, and prostate cancer. This expression pattern suggests that overexpression of Survivin or alterations in survivin gene regulation may commonly occur during tumorigenesis. Survivin is highly expressed in all common human cancers. These observations indicate that apoptosis inhibition may be a general feature of neoplasia.
One of the central functions of apoptosis in maintaining homeostasis is the elimination of damaged and potentially harmful cells. For this process to be effective, the apoptotic machinery must communicate with monitors, or checkpoints, of cell health, sensing DNA damage, adverse environmental conditions, and oncogene or viral transformation. Checkpoint activation under these conditions initiates apoptosis via the assembly of an evolutionarily conserved “apoptosome,” which in mammalian cells comprises an upstream cell-death protease, Caspase-9, the adapter/cofactor protein Apaf-1, mitochondrion-derived cytochrome C and dATP/ATP. Although it is debated how apoptosome assembly promotes Caspase-9 catalytic activity, this process culminates with downstream activation of effector caspases and cleavage of critical cellular substrates. The apoptotic mechanism also appears to monitor cell cycle transitions, assembly of a bipolar mitotic apparatus, the ploidy level of the genome, and the timing of cytokinesis. In this context, dysregulated expression of apoptosis inhibitors Bcl-2 and Bcl-XL has been shown to restrain S phase entry, to promote cell cycle exit, and to cause aneuploidy, further demonstrating a role for the apoptotic machinery in cell-cycle progression.
The IAPs, or inhibitor-of-apoptosis proteins, may be regarded as functional antagonists to a class of proteins known as Apoptosis-Inducing Proteins, or AIPs. One member of the latter class of proteins, Granzyme B, is a serine protease primarily found in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells. Granzyme B plays an important role in inducing apoptotic changes in target cells by cytotoxic cell-mediated killing. Granzyme B is normally produced by natural killer (NK) cells and cytotoxic T-lymphocytes (CTLs) and is released from intracellular granules in response to stimuli that include viral or bacterial infection, abnormally proliferating cells or foreign cell invasion (11, 12). This mechanism protects the host cell from destruction by intracellular pathogens, tumors and foreign cells within the context of the normal immune system (11). Granzyme B is synthesized as a preproenzyme that is activated by two proteolytic cleavages that release an 18-amino-acid leader sequence encoded by exon 1 and a di-peptide motif (Gly-Glu) at the N-terminus. These cleavages are apparently required for full maturation of the enzyme and to allow it to fold into its catalytically active conformation. Although the active form of Granzyme B is generally viewed as that part of the full-length amino acid sequence on the C-terminal side of the di-peptide (GE) processing site, active forms may be considered to include the GE di-peptide and may further include an N-terminal methionine.
Like the caspases, Granzyme B recognizes substrates specifically at aspartic acid for cleavage. (See U.S. Pat. No. 6,537,784, incorporated herein by reference.) To gain entry into its target cell, Granzyme B relies predominantly on perforin, a pore-forming auxiliary protein. Upon entry, active Granzyme B induces apoptosis through both mitochondrial-dependent and mitochondrial-independent mechanisms (11, 13-15). A decrease in mitochondrial membrane potential, direct cleavage of nuclear proteins leading to DNA fragmentation and activation of the Caspase-3 pathway are all known effects of Granzyme B activation (11, 14-16). Granzyme B is known to catalyze cleavage and activation of several caspases, and it is also known to be involved in caspase-independent pathways (see FIG. 1). These diverse mechanisms of Granzyme-B-mediated programmed cell death ensure the successful progression of granule-mediated cell death even in target cells lacking functional caspase proteins, thus providing the host with overlapping safeguards against foreign invaders (16). Despite these sophisticated defense mechanisms, tumor cells have developed molecular evasion mechanisms against Granzyme B-mediated apoptosis (17, 18). These mechanisms include tumor-induced T-cell deletion (by suicide and fratricide), defects in tumor-infiltrating lymphocytes, and impaired presentation of tumor-associated antigens, among others. Recently, certain serpins that inactivate Granzyme-B have also been described (19, 20). It is unknown, however, whether these enzymes are clinically relevant inhibitors Granzyme B in vivo.
Thus, modulation or control of apoptosis provides an alternative route to the use of cytotoxic chemicals or radiation in facilitating the death of deleterious cells, such as cancer cells. Still, a need persists in the art for methods of specifically treating cancer in a manner that does not introduce deleterious side effects typically associated with radiotherapy, chemotherapy and combinational therapies, yet is versatile in exhibiting activity against a range of cancers and is cost-effective in providing a single approach, or set of approaches, to such treatments.